Biologists and clinicians are constantly faced with the problem of assessing tissue damage; currently available methods are cumbersome and often not accurate. Pathologists devote a great deal of time to examining the histology of tissues, counting cells, and estimating tissue damage from the subjective appearance of cells. Radiologists spend a majority of their time trying to find pathological changes on X-rays or magnetic resonance images which would indicate tissue damage. Surgeons rely primarily on visual clues to judge what tissues to remove and what tissues to save, which is not an accurate method of assessing damage, as living tissues often look dead. Additionally, surgeons currently cannot be certain that all diseased tissue is removed during a surgical procedure, which presents a great problem when malignant tissue is being removed.
Even in the laboratory, there is no reliable method for quantifying tissue damage. Investigators spend millions of dollars on tracers and spend months counting cells to measure tissue damage. In order to save time, many investigators simply measure gross areas of necrosis. Since it is not always possible to determine visually which cells are living and which cells are dead, nor the extent of tissue damage to an organ, the currently available methods can be described as crude, at best.
The relative sizes of extracellular and intracellular space in tissue is a valid estimation of the amount of tissue damage in the total space measured. Normal brain tissue, for example, generally has an intracellular volume fraction of about 90%. If tissue is damaged, the intracellular compartments of the dead cells equilibrate with the extracellular space and thus the intracellular volume fraction will drop. The smaller the intracellular volume fraction, the greater the amount of damage in the sample.
The relative sizes of extracellular and intracellular space in brain tissues have been the focus of many scientific studies. Many ingenious methods have been devised to make this determination. In the 1960's, two methods were dominant. One method involved passing an electric current through brain tissues. Because the cells are relatively impervious to electrical currents, the impedance of the brain tissues gave a rough indication of the relative size of the extracellular space. The second method involved the use of macromolecular tracers, including soaking tissue in solutions of these tracers and then measuring the concentration of the substance in the tissue. The ratio of the tissue concentration to the medium was then determined to indicate the space occupied by the tracer.
The tracer methods suffered from several drawbacks. No tracer substance is ideal, and all tracers penetrate into the cells to some degree. Different tracers yield different values of extracellular space. Delivery of the tracer into the tissue is problematical. If the tracer is administered intravenously, its penetration into the brain tissues may be limited by the blood-brain barrier. Blood flow also influences delivery. Therefore, to interpret the data, multiple tracers are necessary: one to monitor blood-brain barrier breakdown, one to measure blood flow, and one to assess extracellular space.
C. Nicholson at New York University developed a method for measuring extracellular volume fraction (V.sub.e /V.sub.t) by introducing a tracer substance such as tetra-ethylammonium (TEA), which should not penetrate the cells. If the extracellular volume increases or decreases, the concentration of TEA changes. By measuring the concentration of TEA with microelectrodes, it is possible to estimate V.sub.e /V.sub.t. The intracellular volume, V.sub.i, divided by V.sub.t, is equal to 1-V.sub.e /V.sub.t. This method also has several major disadvantages. First, it is quite difficult to introduce tracer substances into tissues. The tracer concentration in extracellular space must first be measured before a given injurious event, and then the change must be observed. It is difficult to ensure that the same amount of tracer is injected into the tissue. Second, the method gives the extracellular volume fraction only in the immediate vicinity of the microelectrode recording. Since the ratio of the extracellular volume to the tissue volume may vary within the tissue, it is necessary to sample many points of tissue in order to obtain a representative average value. Thirdly, the ion-selective microelectrodes required to measure the tracer concentrations are fragile and difficult to make. Fourthly, the equations for calculating the extracellular volume fraction require a factor called tortuosity, the convolutions of the pathways through which the ions must diffuse. This factor is resolvable by assuming that the tissue is anisotropic, i.e., that the diffusion of ions is the same in all directions. This method is not accurate when applied to tissues with oriented cellular structures, such as white matter. Because of the complexity of this method, the measured values must be checked very carefully, and errors can very easily arise.
Accordingly, a relatively simple and accurate method of determining intracellular volume fraction has been long sought in order to quantify the extent of tissue damage in any given tissue sample.
Another problem facing biologists and clinicians is obtaining accurate imaging of different types of soft tissue. Existing imaging technology, such as x-ray and NMR, depend on tissue density or differences in proton relaxation rates to identify different tissue types. Both tissue density and proton concentrations have one major drawback. Neither directly reflect tissue damage or biological changes in the tissue. For example, tissue density to x-rays depends on the concentration of x-ray absorbing substances, such as calcium. Calcium concentrations do not change much in acutely injured soft tissues and, if they do change, take place over a period of days, weeks, or months. X-rays, therefore, are primarily useful for visualizing bone and detecting chronic soft tissue injuries. Likewise, magnetic resonance signals resulting from proton relaxation times in tissues represent complex and yet poorly understood contributions from hydrogen in water and organic substances. While magnetic resonance signals do change relatively rapidly in injured tissue, thus allowing early detection and imaging of tissue changes, the nature of the changes is not well understood. Furthermore, the signal changes are relatively small and not necessarily linearly related to known tissue variables. Thus, accurate imaging and differentiation of tissue damage, tumors, and normal tissues of different organs are limited with current technology.
The measurement of neural activity is a very important area of brain research. Presently, this must be done with external or implanted electrodes. There are many disadvantages to such techniques. A non-invasive technique for imaging changes in neural activity in different portions of the brain would be an invaluable research tool.
Probably the most common blood test utilized by clinicians and others is the hematocrit or measurement of cell volume in blood. The present techniques for measuring hematocrit are relatively crude and involve centrifugation of whole blood in a glass capillary tube until the cells are packed at the bottom. The ratio of the cells to total blood volume represents the hematocrit. Most instruments measure hematocrit by optically measuring the heights of the different phases of centrifuged blood. Several disadvantages are associated with this procedure for measuring hematocrits.
1. The instruments required for centrifugation of the capillary tubes and optical measurements tend to be bulky and cannot be easily run on battery. Therefore, portable hematocrit machines are not readily available.
2. The accuracy of hematocrits depends critically on the absence of blood clotting and lysis. If the blood cells were to clot or lyse, the hematocrit becomes grossly inaccurate. Thus, it is essential that the blood be relatively fresh, be placed in a container immediately with anticoagulants, and be centrifuged under appropriate conditions.
3. Hematocrits are relatively imprecise. Most hematocrits measured by eye, using a modified ruler scale, probably are no more accurate than .+-.5% of the mean, e.g., 2% of the normal 40% hematocrit. Factors such as centrifugation force and time, packing of the cells, etc., also may play a role in the variability of measurements.
A quick and accurate test for determining hematocrit in any blood sample, including clotted blood, lysed blood and even blood from corpses, which can be implemented with a small portable device, would be a very valuable addition to the hematological analytical array.